Adrenomedullin to guide therapy of blood pressure decline

ABSTRACT

Subject matter of the present invention is an in vitro method for identifying a subject in need of administration of fluid resuscitation or a vasopressor comprising the following steps:
         Determining the level of proADM and/or fragments thereof having at least 6 amino acids in a bodily fluid of said subject   Correlating said level with the need of said patient for fluid resuscitation or administration of a vasopressor wherein said patient is identified as having such a need if the level of proADM and/or fragments thereof having at least 6 amino acids in the bodily fluid of said subject is above a threshold.

Subject matter of the present invention is an in vitro method foridentifying a subject in need of administration of fluid resuscitationor a vasopressor comprising the following steps:

-   -   Determining the level of proADM and/or fragments thereof having        at least 6 amino acids in a bodily fluid of said subject    -   Correlating said level with the need of said patient for fluid        resuscitation or administration of a vasopressor wherein said        patient is identified as having such a need if the level of        proADM and/or fragments thereof having at least 6 amino acids in        the bodily fluid of said subject is above a threshold.

The peptide adrenomedullin (ADM) was described for the first time inKitamura et al., (cf. 1; numerical data are based on the attached listof references) as a novel hypotensive peptide comprising 52 amino acids,which had been isolated from a human pheochromocytoma. In the same year,cDNA coding for a precursor peptide comprising 185 amino acids and thecomplete amino acid sequence of this precursor peptide were alsodescribed. The precursor peptide, which comprises, inter alia, a signalsequence of 21 amino acids at the N-terminus, is referred to as“preproadrenomedullin” (pre-proADM). Pre-proADM comprises 185 aminoacids. The mature ADM is displayed in SEQ ID No. 4 and ADM-Gly isdisplayed in SEQ No. 5.

The mature peptide adrenomedullin (ADM) is an amidated peptide whichcomprises 52 amino acids (SEQ ID No: 4) and which comprises the aminoacids 95 to 146 of pre-proADM, from which it is formed by proteolyticcleavage. To date, substantially only a few fragments of the peptidefragments formed in the cleavage of the pre-proADM have been moreexactly characterized, in particular the physiologically active peptidesadrenomedullin (ADM) and “PAMP”, a peptide comprising 20 amino acids(22-41) which follows the 21 amino acids of the signal peptide inpre-proADM. For both ADM and PAMP, physiologically active sub-fragmentshave furthermore been discovered and investigated in more detail. Thediscovery and characterization of ADM in 1993 triggered intensiveresearch activity and a flood of publications, the results of which haverecently been summarized in various review articles, in the context ofthe present description, reference being made in particular to thearticles to be found in an issue of “Peptides” devoted to ADM (Peptides22 (2001)), in particular (2) and (3). A further review is (4). In thescientific investigations to date, it has been found, inter alia, thatADM may be regarded as a polyfunctional regulatory peptide. It isreleased into the circulation in an inactive form extended by glycine(5). There is also a binding protein (6) which is specific for ADM andprobably likewise modulates the effect of ADM.

Those physiological effects of ADM as well as of PAMP which are ofprimary importance in the investigations to date were the effectsinfluencing blood pressure. Thus, ADM is an effective vasodilator, itbeing possible to associate the hypotensive effect with in particularpeptide segments in the C-terminal part of ADM.

It has furthermore been found that the above-mentioned furtherphysiologically active peptide PAMP formed from pre-proADM likewiseexhibits a hypotensive effect, even if it appears to have an actionmechanism differing from that of ADM (cf. in addition to theabove-mentioned review articles (3) and (4) also (7), (8) or (9) and(10)).

It has furthermore been found that the concentrations of ADM which canbe measured in the circulation and other biological fluids are, in anumber of pathological states, significantly above the concentrations tobe found in healthy control persons. Thus, the ADM level in patientswith congestive heart failure, myocardial infarction, kidney diseases,hypertensive disorders, Diabetes mellitus, in the acute phase of shockand in sepsis and septic shock are significantly increased, although todifferent extents. The PAMP concentrations are also increased in some ofsaid pathological states, but the plasma levels are reduced relative toADM ((3); page 1702).

It is furthermore known that unusually high concentrations of ADM are tobe observed in sepsis or in septic shock (cf. (3) and (11), (12), (13),(14) and (15)). The findings are related to the typical hemodynamicchanges which are known as typical phenomena of the course of a diseasein patients with sepsis and other severe syndromes, such as, forexample, SIRS.

Although it is assumed that ADM and PAMP are formed from the sameprecursor peptide, pre-proADM, in which the amino acid sequencescorresponding to these peptides are present as partial peptides inequimolar amounts, the concentrations of ADM or PAMP measurable inbiological fluids apparently differ. This is nothing unusual.

Thus, the measurable concentrations of different degradation products ofone and the same precursor peptide may be different, for example,because they are the result of different competing degradation pathwayswhich, for example in the case of different pathological states, lead todifferent fragmentation of a precursor peptide and hence to differentdegradation products. Certain partial peptides contained in theprecursor peptide may be formed as free peptides or may not be formed,and/or different peptides are formed in different ways and in differentamounts. Even if only a single degradation pathway is taken forprocessing a precursor peptide, and hence all degradation productsoriginate from one and the same precursor peptide and must have beenformed per se primarily in equimolar amounts, the steady stateconcentrations of different partial peptides and fragments measurable inbiological fluids may be very different, namely, for example, whenindividual ones thereof are formed at a different rate and/or havedifferent individual stabilities (lifetimes) in the respectivebiological fluid, or if they are removed from circulation on the basisof different clearance mechanisms and/or at different clearance rates.

Adrenomedullin plays pivotal roles during sepsis development ((16),(17)) and in numerous acute and chronic diseases ((18), (4)).

Several methods have been described to measure circulating levels ofADM: Either ADM directly or indirectly by determining a more stablefragment of its cognate precursor peptide. Very recently a method hasbeen published describing an assay to measure circulating mature ADM (DiSomma S, Magrini L, Travaglino F, Lalle I, Fiotti N, Cervellin G, AvanziG C, Lupia E, Maisel A, Hein F et al: Opinion paper on innovativeapproach of biomarkers for infectious diseases and sepsis management inthe emergency department. Clinical chemistry and laboratory medicine:CCLM/FESCC 2013:1-9.)

Other methods to quantify fragments derived from the ADM precursor havebeen described, e.g. the measurement of MR-proADM (Morgenthaler N G,Struck J, Alonso C, Bergmann A.Clin Chem. 2005 October; 51(10):1823-9.),PAMP (Washimine H, Kitamura K, Ichiki Y, Yamamoto Y, Kangawa K, MatsuoH, Eto T. Biochem Biophys Res Commun. 1994 Jul. 29; 202(2):1081-7.),CT-proADM (EP211552). A commercial assay for the measurement ofMR-proADM is available (BRAHMS MR-proADM KRYPTOR; BRAHMS GmbH,Hennigsdorf, Germany) (Clin Biochem. 2009 May; 42(7-8):725-8. doi:10.1016/j.clinbiochem.2009.01.002. Epub 2009 Jan 23).

Homogeneous time-resolved fluoroimmunoassay for the measurement ofmidregional proadrenomedullin in plasma on the fully automated systemB.R.A.H.M.S KRYPTOR (Caruhel P, Mazier C, Kunde J, Morgenthaler N G,Darbouret B.). As these peptides are generated in a stoichiometric ratiofrom the same precursor, their plasma levels are correlated to a certainextent.

Only in a few studies plasma ADM has been measured in patients withsystemic inflammation, sepsis, severe sepsis or septic shock, and levelshave been correlated with hemodynamic parameters:

In a study by Hirata et al. plasma ADM in septic patients was found tobe correlated with heart rate, right arterial pressure, but not withmean arterial pressure (MAP) (Hirata Y, Mitaka C, Sato K, Nagura T,Tsunoda Y, Amaha K, Marumo F: Increased circulating adrenomedullin, anovel vasodilatory peptide, in sepsis. The Journal of clinicalendocrinology and metabolism 1996, 81(4):1449-1453).

Nishio et al. reported that increased plasma concentrations of ADM werecorrelated with relaxation of vascular tone in patients with septicshock (correlation with cardiac index, stroke volume index, heart rate,decrease in diastolic blood pressure, systemic vascular resistance indexand pulmonary vascular resistance index), however there was nosignificant correlation with mean blood pressure [19].

In healthy subjects under exercise a significant negative correlation ofplasma ADM and MAP was found [20].

Nothing is known about the association of circulating ADM or relatedpeptide levels as pro-ADM or fragments thereof and the requirements forfluid resuscitation and vasopressors in patients developing shock. Thisis an unmet medical need as vasopressors are usually given very latewhen the condition of the patient is very serious. It is an unmetmedical need to identify those patients in need of fluid resuscitationand vasopressors before the condition of the patient is very serious. Itis an unmet medical need to predict the requirement for fluidresuscitation and vasopressor therapy earlier than by blood pressuremeasurement applying the cut-off value of 65 mm Hg, as recommended inthe guidelines [21]. If blood pressure fells, this leads to diminishedoxygen supply, organ dysfunction and death. It is thus an unmet need toearly identify patients who are at risk to develop a blood pressuredecline. If such patients could be identified earlier, other, e.g.higher cut-off values for mean arterial pressure could be applied toinitiate fluid resuscitation and vasopressor therapy. Higherthreshold-levels are <70 mm Hg, preferably <75 mm Hg. This means thattherapy starts already above 65 mmHg

Subject matter of the present invention is an in vitro method foridentifying a subject in need of fluid resuscitation or administrationof a vasopressor comprising the following steps:

-   -   Determining the level of proADM and/or fragments thereof having        at least 6 amino acids in a bodily fluid of said subject    -   Correlating said level with the need of said subject or patient        for fluid resuscitation or administration of a vasopressor        wherein said subject or patient is identified as having such a        need if the level of proADM and/or fragments thereof having at        least 6 amino acids in the bodily fluid of said subject is above        a threshold.

In one embodiment of the methods according to the invention the subjecthas a mean arterial pressure >65 mm Hg. Further, said subject may have amean arterial pressure <75 mmg Hg, in another embodiment <70 mmg Hg.

In one embodiment of the invention said method is a method for the earlyidentification of fluid resuscitation or administration of a vasopressorwherein earlier means earlier than by blood pressure measurementapplying a cut-off value of 65 mmg Hg or before the blood pressuredropped to 65 mmg Hg.

A bodily fluid according to the present invention is in one particularembodiment a blood sample. A blood sample may be selected from the groupcomprising whole blood, serum and plasma.

In a specific embodiment of the invention said proADM and/or fragmentsthereof having at least 6 amino acids is/are selected from the groupcomprising:

(proADM): 164 amino acids (22-185 of preproADM) SEQ ID No. 1ARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVKAGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMNNFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSKISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL(Proadrenomedullin N-20 terminal peptide): Peptides 22-41 SEQ ID No. 2ARLDVASEF RKKWNKWALS R (MidregionalproAdrenomedullin, MR-proADM):Peptides45-92 SEQ ID No. 3ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV(mature Adrenomedullin (mature ADM); amidated): Peptides 95-146-CONH₂SEQ ID No. 4 YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSKISPQGY-CONH₂ (Adrenomedullin 1-52-Gly (ADM 1-52-Gly)): Peptides 95-147SEQ ID No. 5 YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYG(C-terminal proAdrenomedullin, CT-proADM): Peptides 148-185 SEQ ID No. 6RRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

In a specific embodiment of the invention said proADM and/or fragmentsthereof having at least 6 amino acids is/are selected from the groupcomprising mature ADM (SEQ ID No. 4) and/or mature ADM 1-52-Gly (SEQ IDNo. 5) and MR-proADM (SEQ ID No. 3) and CT-proADM (SEQ ID No. 6).

In a specific embodiment of the invention either the level of mature ADM(SEQ ID No. 4) and/or mature ADM 1-52-Gly (SEQ ID No.5)—immunoreactivity or the level of MR-proADM (SEQ ID No. 3)immunoreactivity or the level of CT-proADM (SEQ ID No. 6)immunoreactivity is determined and correlated with the need of saidpatient for fluid resuscitation or administration of a vasopressorwherein said patient is identified as having such a need if the level ofmature ADM (SEQ ID No. 4) and/or mature ADM 1-52-Gly (SEQ ID No.5)—immunoreactivity or the level of MR-proADM (SEQ ID No. 3)immunoreactivity or the level of CT-proADM (SEQ ID No. 6)immunoreactivity in the bodily fluid of said subject is above athreshold.

In a specific embodiment of the invention the level of pro-ADM and/orfragments thereof is determined by using at least one binder selectedfrom the group: a binder that binds to a region comprised within thefollowing sequence of mature ADM (SEQ ID No. 4) and/or mature ADM1-52-Gly (SEQ ID No. 5) and a second binder that binds to a regioncomprised within the sequence of mature ADM (SEQ ID NO. 4) and/or matureADM 1-52-Gly (SEQ ID No. 5).

In a specific embodiment of the invention the level of pro-ADM and/orfragments thereof is determined by using at least one binder selectedfrom the group: a binder that binds to a region comprised within thesequence of MR-proADM (SEQ ID No. 3) and a second binder that binds to aregion comprised within the sequence of MR-proADM (SEQ ID No. 3)

In a specific embodiment of the invention the level of pro-ADM and/orfragments thereof is determined by using at least one binder selectedfrom the group: a binder that binds to a region comprised within thesequence of CT-proADM (SEQ ID No. 6) and a second binder that binds to aregion comprised within the sequence of CT-pro ADM (SEQ ID No. 6).

In a specific embodiment of the invention an assay is used fordetermining the level of proADM and/or fragments thereof having at least6 amino acids wherein the assay sensitivity of said assay is able toquantify the ADM of healthy subjects and is <70 pg/ml, preferably <40pg/ml and more preferably <10 pg/ml.

In a specific embodiment of the invention said binder exhibits anbinding affinity to proADM and/or fragments thereof of at least 10⁷ M⁻¹,preferred 10⁸ M⁻¹, preferred affinity is greater than 10⁹ M⁻¹, mostpreferred greater than 10¹⁰ M⁻¹ A person skilled in the art knows thatit may be considered to compensate lower affinity by applying a higherdose of compounds and this measure would not lead out-of-the-scope ofthe invention.

To determine the affinity of the antibodies to Adrenomedullin, thekinetics of binding of Adrenomedullin to immobilized antibody wasdetermined by means of label-free surface plasmon resonance using aBiacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany).Reversible immobilization of the antibodies was performed using ananti-mouse Fc antibody covalently coupled in high density to a CM5sensor surface according to the manufacturer's instructions (mouseantibody capture kit; GE Healthcare), (22).

In a specific embodiment of the invention said binder is selected fromthe group comprising an antibody or an antibody fragment or a non-Igscaffold binding to proADM and/or fragments thereof.

In a specific embodiment of the invention an assay is used fordetermining the level of proADM and/or fragments thereof having at least6 amino acids wherein such assay is a sandwich assay, preferably a fullyautomated assay.

In one embodiment of the invention it may be a so-called POC-test(point-of-care) that is a test technology which allows performing thetest within less than 1 hour near the patient without the requirement ofa fully automated assay system. One example for this technology is theimmunochromatographic test technology.

In one embodiment of the invention such an assay is a sandwichimmunoassay using any kind of detection technology including but notrestricted to enzyme label, chemiluminescence label,electrochemiluminescence label, preferably a fully automated assay. Inone embodiment of the invention such an assay is an enzyme labeledsandwich assay. Examples of automated or fully automated assay compriseassays that may be used for one of the following systems: RocheElecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®,BiomerieuxVidas®, Alere Triage®.

A variety of immunoassays are known and may be used for the assays andmethods of the present invention, these include: radioimmunoassays(“RIA”), homogeneous enzyme-multiplied immunoassays (“EMIT”), enzymelinked immunoadsorbent assays (“ELISA”), apoenzyme reactivationimmunoassay (“ARIS”), dipstick immunoassays and immuno-chromotographyassays.

In a specific embodiment of the invention at least one of said twobinders is labeled in order to be detected.

The preferred detection methods comprise immunoassays in various formatssuch as for instance radioimmunoassay (RIA), chemiluminescence- andfluorescence-immunoassays, Enzyme-linked immunoassays (ELISA),Luminex-based bead arrays, protein microarray assays, and rapid testformats such as for instance immunochromatographic strip tests.

In a preferred embodiment said label is selected from the groupcomprising chemiluminescent label, enzyme label, fluorescence label,radioiodine label.

The assays can be homogenous or heterogeneous assays, competitive andnon-competitive assays. In one embodiment, the assay is in the form of asandwich assay, which is a non-competitive immunoassay, wherein themolecule to be detected and/or quantified is bound to a first antibodyand to a second antibody. The first antibody may be bound to a solidphase, e.g. a bead, a surface of a well or other container, a chip or astrip, and the second antibody is an antibody which is labeled, e.g.with a dye, with a radioisotope, or a reactive or catalytically activemoiety. The amount of labeled antibody bound to the analyte is thenmeasured by an appropriate method. The general composition andprocedures involved with “sandwich assays” are well-established andknown to the skilled person (23).

In another embodiment the assay comprises two capture molecules,preferably antibodies which are both present as dispersions in a liquidreaction mixture, wherein a first labelling component is attached to thefirst capture molecule, wherein said first labelling component is partof a labelling system based on fluorescence- orchemiluminescence-quenching or amplification, and a second labellingcomponent of said marking system is attached to the second capturemolecule, so that upon binding of both capture molecules to the analytea measurable signal is generated that allows for the detection of theformed sandwich complexes in the solution comprising the sample.

In another embodiment, said labeling system comprises rare earthcryptates or rare earth chelates in combination with fluorescence dye orchemiluminescence dye, in particular a dye of the cyanine type.

In the context of the present invention, fluorescence based assayscomprise the use of dyes, which may for instance be selected from thegroup comprising FAM (5-or 6-carboxyfluorescein), VIC, NED, Fluorescein,Fluoresceinisothiocyanate (FITC), IRD-700/800, Cyanine dyes, such asCY3, CYS, CY3.5, CY5.5, Cy7, Xanthen,6-Carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), TET,6-Carboxy-4′,5′-dichloro-2′,7′-dimethodyfluorescein (JOE),N,N,N′,N′-Tetramethyl-6-carboxyrhodamine (TAMRA), 6-Carboxy-X-rhodamine(ROX), 5-Carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6),Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes,such as BODIPY TMR, Oregon Green, Coumarins such as Umbelliferone,Benzimides, such as Hoechst 33258; Phenanthridines, such as Texas Red,Yakima Yellow, Alexa Fluor, PET, Ethidiumbromide, Acridinium dyes,Carbazol dyes, Phenoxazine dyes, Porphyrine dyes, Polymethin dyes, andthe like.

In the context of the present invention, chemiluminescence based assayscomprise the use of dyes, based on the physical principles described forchemiluminescent materials in (24). Preferred chemiluminescent dyes areacridiniumesters.

As mentioned herein, an “assay” or “diagnostic assay” can be of any typeapplied in the field of diagnostics. Such an assay may be based on thebinding of an analyte to be detected to one or more capture probes witha certain affinity. Concerning the interaction between capture moleculesand target molecules or molecules of interest, the affinity constant ispreferably greater than 10⁸ M⁻¹.

In the context of the present invention, “binder molecules” aremolecules which may be used to bind target molecules or molecules ofinterest, i.e. analytes (i.e. in the context of the present inventionPCT and fragments thereof), from a sample. Binder molecules must thus beshaped adequately, both spatially and in terms of surface features, suchas surface charge, hydrophobicity, hydrophilicity, presence or absenceof lewis donors and/or acceptors, to specifically bind the targetmolecules or molecules of interest. Hereby, the binding may for instancebe mediated by ionic, van-der-Waals, pi-pi, sigma-pi, hydrophobic orhydrogen bond interactions or a combination of two or more of theaforementioned interactions between the capture molecules and the targetmolecules or molecules of interest. In the context of the presentinvention, binder molecules may for instance be selected from the groupcomprising a nucleic acid molecule, a carbohydrate molecule, a PNAmolecule, a protein, an antibody, a peptide or a glycoprotein.Preferably, the binder molecules are antibodies, including fragmentsthereof with sufficient affinity to a target or molecule of interest,and including recombinant antibodies or recombinant antibody fragments,as well as chemically and/or biochemically modified derivatives of saidantibodies or fragments derived from the variant chain with a length ofat least 12 amino acids thereof.

Chemiluminescent label may be acridinium ester label, steroid labelsinvolving isoluminol labels and the like.

Enzyme labels may be lactate dehydrogenase (LDH),creatinekinase (CPK),alkaline phosphatase, aspartateaminotransferace (AST), alanineaminotransferace (ALT),acid phosphatase, glucose-6-phosphatedehydrogenase and so on.

In one embodiment of the invention at least one of said two binders isbound to a solid phase as magnetic particles, and polystyrene surfaces.

In a specific embodiment of the invention at least one of said twobinders is bound to a solid phase.

In one embodiment of the invention the concentration of ADM or fragmentsthereof measured in the sample is in the range between 10-500 pg/ml inplasma or blood.

The ADM levels of the present invention or proADM levels or fragmentsthereof, respectively, have been determined with the described ADMassay, as outlined in the examples (or proADM or fragments thereofassays, respectively). The above mentioned values might be different inother ADM assays (or proADM or fragments thereof assays, respectively),depending upon their way of calibration. The above mentioned valuesshall apply for such differently calibrated ADM assays accordingly,taking into account the differences in calibration. ADM assays could becalibrated by correlation and adjustment via their normal ranges(healthy population). (or proADM or fragments thereof assays,respectively) Alternatively, commercially available control samplescould be used for adjustment of different calibrations (ICI Diagnostics,Berlin, Germany)

With the described ADM assay, the median of of a normal population hasbeen determined to be 24.7 pg/mL.

In a specific embodiment of the invention a threshold for plasma ADM of90 pg/ml, preferably 70 pg/ml is applied.

In a specific embodiment of the invention a threshold for plasmaMR-proADM of 0.9 nmol/L, preferably 0.7 nmol/L is applied

In a specific embodiment of the invention a threshold for plasmaCT-proADM of 1.0 nmol/L, preferably 0.8 nmol/L is applied.

If the level of plasma ADM or plasma MR-proADM or plasma CT-proADM isabove said threshold the person might be in need of treatment with avasopressor.

In a specific embodiment of the invention said sample is selected fromthe group comprising human citrate plasma, heparin plasma, EDTA plasma,whole blood.

The subject that may be in need of fluid resuscitation or treatment witha vasopressor may suffer from a condition selected from the groupcomprising: patients at risk to develop physiological shock states, asdescribed in more detail below, but also infections, SIRS, sepsis, heartfailure, cardiopulmonary arrest, postoperative cardiac surgery, rightventricular infarction, bradyarrhythmias, polytrauma, burns, kidneyinjury.

This type of shock can be caused by:

-   -   Severe bleeding.    -   Pulmonary embolus (a blood clot in the lungs).    -   Severe vomiting and diarrhoea.    -   Spinal injury.    -   Poisoning.

There are also specific types of physiological shock, with veryparticular symptoms.

Cardiogenic shock:

Cardiogenic shock occurs when the heart is severely damaged—by a majorheart attack, for example—and is no longer able to pump blood around thebody properly, causing very low blood pressure. This develops afterabout eight per cent of heart attacks. It can be difficult to treat, butdrugs may be given to make the heart beat stronger. This may be enoughto bring someone through the worst until the heart can mend itself, butcardiogenic shock is still fatal in as many as eight out of ten cases.New treatments to ‘revascularise’ or restore blood flow to the heartmuscle are improving survival rates.

Septic shock:

This occurs when an overwhelming bacterial infection causes bloodpressure to drop. It's fatal in more than 50% of cases. Although it'scaused by bacterial infection, treating septic shock with antibiotics isfar from simple, because the bacteria release massive amounts of toxinwhen they are killed off, which initially makes the shock worse. It mustalways be treated in hospital where the correct drugs and fluid supportcan be given. One type of septic shock is toxic shock syndrome—a rarebut severe illness caused by certain strains of the bacteriaStaphylococcus aureus.

Anaphylactic shock:

Anaphylactic shock is a severe allergic reaction. Common triggersinclude bee and wasp stings, nuts, shellfish, eggs, latex and certainmedications, including penicillin. Symptoms include:

-   -   Burning and swelling of the lips and tongue.    -   Difficulty breathing (like in an asthma attack).    -   Red, itchy or blistered skin, sneezing.    -   Watery eyes.    -   Nausea.    -   Anxiety.

Anaphylaxis requires urgent treatment in hospital. People at risk shouldalways carry an emergency anaphylaxis treatment kit that includesadrenaline.

Sepsis and its escalated forms (severe sepsis, septic shock) continue tobe a major medical problem, with mortality rates ranging from 30 to 70%.Despite advances in supportive care, each year 750,000 people developsepsis and 225,000 die in the United States alone, and the incidence ofsepsis is rising at rates between 1.5% and 8% per year [4-6]. In orderto save the life of a septic patient, it is essential to first timelyfight the infectious stimulus by antibiotics or other measures, andsecond, to timely recognize escalation of the situation, e.g. whensevere sepsis proceeds to septic shock, because only then suitablevasopressor therapy can be initiated early. Any delay would increase therisk of the patient to die.

The conditions under which it is recommended to initiate a fluidresuscitation or vasopressor/inotrope therapy in patients progressing toseptic shock are described in the guidelines of the Surviving SepsisCampaign [3]: It is recommended to apply vasopressors for hypotensionthat does not respond to initial fluid resuscitation to maintain a meanarterial pressure (MAP) of ≥65 mm Hg. The guideline also talks aboutwhich vasopressor/inotrope to apply preferentially when. The currentconsensus view is: Norepinephrine as the first choice vasopressor.Epinephrine (added to and potentially substituted for norepinephrine)when an additional agent is needed to maintain adequate blood pressure.Vasopressin 0.03 units/minute can be added to norepinephrine (NE) withintent of either raising MAP or decreasing NE dosage (UG). In general,currently used vasopressors and inotropes in clinical practice are [7,8]:

Catecholamines (Dopamine, Dobutamine, Norepinephrine, Epinephrine,Isoproterenol, Phenylephrine), Phosphodiesterase III inhibitors(Milrinone, Amrinone), Vasopressin, Levosimendan.

Additionally, other vasoactive compounds are under development, such asfor instance Selepressin, a selective vasopressin V la receptor agonist[9] and anti-Adrenomedullin antibodies.

Other compounds have been investigated, but clinical data available forthese treatments are sparse, and fairly equivocal results of theseapproaches have been obtained in larger trials [10]. These areinhibitors of ATP-dependent K+-channels (glibenclamide; [11, 12]) of NOS(N^(G)-monomethyl-L-arginine [13, 14]) and of cGMP (methylene blue [15,16]).

Vasopressors and inotropes are clinically applied to treat and prevendvarious physiological shock types, but also cardiovascular diseases(congestive heart failure, cardiopulmonary arrest, postoperative cardiacsurgery, right ventricular infarction, bradyarrhythmias) [7]

Since blood pressure is always monitored in patients that present in acritical condition, from a clinical point of view, patients with valuesabove the respective thresholds e.g. high ADM (>70 pg/ml) withoutvasopressor need at presentation should be vasopressor treated byadapting the points of decision from <66 mmHg MAP to e.g. <75 mmHgaiming earlier support of circulation to protect patient from low bloodpressure associated organ dysfunctions and subsequent high mortality.Using this rule for patients >70 pg/ml ADM and treating withvasopressors at MAP <=75 mmHg, patients (Group 3) would be treated inaverage 1,6 days before standard of care treatment (<=66 mmHg).

Thus, in a specific embodiment of the present invention said patient isidentified as having a need of administration of a vasopressor if thelevel of proADM and/or fragments thereof having at least 6 amino acidsin the bodily fluid of said subject is above a threshold and if thepatient has a </=75 mmHg MAP but preferably >66 mmHg, morepreferably >70 mmHg MAP.

In a specific embodiment of the invention a threshold for plasma ADM of90 pg/ml, preferably 70 pg/ml is applied and/or the patient has a <=/75mmHg MAP but preferably >66mmHg, more preferably >70 mmHg MAP.

In a specific embodiment of the invention a threshold for plasmaMR-proADM of 0.9 nmol/L, preferably 0.7 nmol/L is applied and/or thepatient has a </=75 mmHg MAP but preferably >66 mmHg, morepreferably >70 mmHg MAP.

In an specific embodiment of the invention a threshold for plasma CTproADM of 1.0 nmol/L preferably 0.8 nmol/L is applied and/or the patienthas a </=75 mmHg MAP but preferably >66 mmHg, more preferably >70 mmHgMAP.

If the level of plasma ADM or plasma MR-proADM or plasma CT proADM isabove said threshold and/or if the patient has a </=75 mmHg MAP butpreferably >66 mmHg, more preferably >70 mmHg MAP the person might be inneed of treatment with a vasopressor.

Fluid replacement or fluid resuscitation is the medical practice ofreplenishing bodily fluid lost through sweating, bleeding, fluid shiftsor other pathologic processes as above described. Fluids can be replacedvia oral administration (drinking), intravenous administration,rectally, or by hypodermoclysis, the direct injection of fluid into thesubcutaneous tissue. Fluids administered by the oral and hypodermicroutes are absorbed more slowly than those given intravenously. Oralrehydration therapy (ORT) is a simple treatment for dehydrationassociated with diarrhea, particularlygastroenteritis/gastroenteropathy, such as that caused by cholera orrotavirus. ORT consists of a solution of salts and sugars which is takenby mouth.

In severe dehydration, intravenous fluid replacement is preferred, andmay be lifesaving. It is especially useful where there is depletion offluid both in the intracellular space and the vascular spaces.

Fluid replacement is also indicated in fluid depletion due to any of theabove described conditions.

An antibody according to the present invention is a protein includingone or more polypeptides substantially encoded by immunoglobulin genesthat specifically binds an antigen. The recognized immunoglobulin genesinclude the kappa, lambda, alpha (IgA), gamma (IgG₁, IgG₂, IgG₃, IgG₄),delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as wellas the myriad immunoglobulin variable region genes. Full-lengthimmunoglobulin light chains are generally about 25 Kd or 214 amino acidsin length. Full-length immunoglobulin heavy chains are generally about50 Kd or 446 amino acid in length.

Light chains are encoded by a variable region gene at the NH2-terminus(about 110 amino acids in length) and a kappa or lambda constant regiongene at the COOH-terminus. Heavy chains are similarly encoded by avariable region gene (about 116 amino acids in length) and one of theother constant region genes.

The basic structural unit of an antibody is generally a tetramer thatconsists of two identical pairs of immunoglobulin chains, each pairhaving one light and one heavy chain. In each pair, the light and heavychain variable regions bind to an antigen, and the constant regionsmediate effector functions. Immunoglobulins also exist in a variety ofother forms including, for example, Fv, Fab, and (Fab′)₂, as well asbifunctional hybrid antibodies and single chains (e.g., Lanzavecchiaetal., Eur. J. Immunol. 17:105,1987; Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988; Hoodet al., Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller and Hood,Nature 323:15-16,1986). An immunoglobulin light or heavy chain variableregion includes a framework region interrupted by three hypervariableregions, also called complementarity determining regions (CDR's) (see,Sequences of Proteins of Immunological Interest, E. Kabatet al., U.S.Department of Health and Human Services, 1983). As noted above, the CDRsare primarily responsible for binding to an epitope of an antigen. Animmune complex is an antibody, such as a monoclonal antibody, chimericantibody, humanized antibody or human antibody, or functional antibodyfragment, specifically bound to the antigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody can be joined to human constant segments, such askappa and gamma 1 or gamma 3. In one example, a therapeutic chimericantibody is thus a hybrid protein composed of the variable orantigen-binding domain from a mouse antibody and the constant oreffector domain from a human antibody, although other mammalian speciescan be used, or the variable region can be produced by moleculartechniques. Methods of making chimeric antibodies are well known in theart, e.g., see U.S. Pat. No. 5,807,715. A “humanized” immunoglobulin isan immunoglobulin including a human framework region and one or moreCDRs from a non-human (such as a mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor” and the human immunoglobulin providing the framework istermed an “acceptor.” In one embodiment, all the CDRs are from the donorimmunoglobulin in a humanized immunoglobulin. Constant regions need notbe present, but if they are, they must be substantially identical tohuman immunoglobulin constant regions, i.e., at least about 85-90%, suchas about 95% or more identical. Hence, all parts of a humanizedimmunoglobulin, except possibly the CDRs, are substantially identical tocorresponding parts of natural human immunoglobulin sequences. A“humanized antibody” is an antibody comprising a humanized light chainand a humanized heavy chain immunoglobulin. A humanized antibody bindsto the same antigen as the donor antibody that provides the CDRs. Theacceptor framework of a humanized immunoglobulin or antibody may have alimited number of substitutions by amino acids taken from the donorframework. Humanized or other monoc lonal antibodies can have additionalconservative amino acid substitutions which have substantially no effecton antigen binding or other immunoglobulin functions. Exemplaryconservative substitutions are those such as gly, ala; val, ile, leu;asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanizedimmunoglobulins can be constructed by means of genetic engineering(e.g., see U.S. Pat. No. 5,585,089). A human antibody is an antibodywherein the light and heavy chain genes are of human origin. Humanantibodies can be generated using methods known in the art. Humanantibodies can be produced by immortalizing a human B cell secreting theantibody of interest Immortalization can be accomplished, for example,by EBV infection or by fusing a human B cell with a myeloma or hybridomacell to produce a trioma cell. Human antibodies can also be produced byphage display methods (see, e.g., Dower et al., PCT Publication No.WO91/17271; McCafferty et al., PCT Publication No. WO92/001047; andWinter, PCT Publication No. WO92/20791), or selected from a humancombinatorial monoclonal antibody library (see the Morphosys website).Human antibodies can also be prepared by using transgenic animalscarrying a human immunoglobulin gene (for example, see Lonberget al.,PCT Publication No. WO93/12227; and Kucherlapati, PCT Publication No.WO91/10741).

Thus, the antibody may have the formats known in the art. Examples arehuman antibodies, monoclonal antibodies, humanized antibodies, chimericantibodies, CDR-grafted antibodies. In a preferred embodiment antibodiesaccording to the present invention are recombinantly produced antibodiesas e.g. IgG, a typical full-length immunoglobulin, or antibody fragmentscontaining at least the F-variable domain of heavy and/or light chain ase.g. chemically coupled antibodies (fragment antigen binding) includingbut not limited to Fab-fragments including Fab minibodies, single chainFab antibody, monovalent Fab antibody with epitope tags, e.g. Fab-V5Sx2;bivalent Fab (mini-antibody) dimerized with the CH3 domain; bivalent Fabor multivalent Fab, e.g. formed via multimerization with the aid of aheterologous domain, e.g. via dimerization of dHLXdomains, e.g.Fab-dHLX-FSx2; F(ab′)2-fragments, scFv-fragments, multimerizedmultivalent or/and multispecificscFv-fragments, bivalent and/orbispecificdiabodies, BITE® (bispecific T-cell engager), trifunctionalantibodies, polyvalent antibodies, e.g. from a different class than G;single-domain antibodies, e.g. nanobodies derived from camelid or fishimmunoglobulines and numerous others.

In addition to antibodies other biopolymer scaffolds are well known inthe art to complex a target molecule and have been used for thegeneration of highly target specific biopolymers. Examples are aptamers,spiegelmers, anticalins and conotoxins.

In a preferred embodiment the antibody format is selected from the groupcomprising Fv fragment, scFv fragment, Fab fragment, scFab fragment,(Fab)2 fragment and scFv-Fc Fusion protein. In another preferredembodiment the antibody format is selected from the group comprisingscFab fragment, Fab fragment, scFv fragment and bioavailabilityoptimized conjugates thereof, such as PEGylated fragments. One of themost preferred formats is the scFab format.

Non-Ig scaffolds may be protein scaffolds and may be used as antibodymimics as they are capable to bind to ligands or antigenes. Non-Igscaffolds may be selected from the group comprising tetranectin-basednon-Ig scaffolds (e.g. described in US 2010/0028995), fibronectinscaffolds (e.g. described in EP 1266 025; lipocalin-based scaffolds((e.g. described in WO 2011/154420); ubiquitin scaffolds (e.g. describedin WO 2011/073214), transferring scaffolds (e.g. described in US2004/0023334), protein A scaffolds (e.g. described in EP 2231860),ankyrin repeat based scaffolds (e.g. described in WO 2010/060748),microproteins, preferably microproteins forming a cystine knot)scaffolds (e.g. described in EP 2314308), Fyn SH3 domain based scaffolds(e.g. described in WO 2011/023685) EGFR-A-domain based scaffolds (e.g.described in WO 2005/040229) and Kunitz domain based scaffolds (e.g.described in EP 1941867).

In one embodiment of the invention antibodies according to the presentinvention may be produced as follows:

A Balb/c mouse was immunized with ADM-100 μg Peptide-BSA-Conjugate atday 0 and 14 (emulsified in 100 μl complete Freund's adjuvant) and 50 μgat day 21 and 28 (in 100 μl incomplete Freund's adjuvant). Three daysbefore the fusion experiment was performed, the animal received 50 μg ofthe conjugate dissolved in 100 μl saline, given as one intraperitonealand one intravenous injection.

Splenocytes from the immunized mouse and cells of the myeloma cell lineSP2/0 were fused with lml 50% polyethylene glycol for 30s at 37° C.After washing, the cells were seeded in 96-well cell culture plates.Hybrid clones were selected by growing in HAT medium [RPMI 1640 culturemedium supplemented with 20% fetal calf serum and HAT-Supplement]. Aftertwo weeks the HAT medium is replaced with HT Medium for three passagesfollowed by returning to the normal cell culture medium.

The cell culture supernatants were primary screened for antigen specificIgG antibodies three weeks after fusion. The positive testedmicrocultures were transferred into 24-well plates for propagation.After retesting, the selected cultures were cloned and recloned usingthe limiting-dilution technique and the isotypes were determined (seealso Lane, R. D. (1985). A short-duration polyethylene glycol fusiontechnique for increasing production of monoclonal antibody-secretinghybridomas. J. Immunol. Meth. 81: 223-228; Ziegler, B. et al. (1996)Glutamate decarboxylase (GAD) is not detectable on the surface of ratislet cells examined by cytofluorometry and complement-dependentantibody-mediated cytotoxicity of monoclonal GAD antibodies, Horm.Metab. Res. 28: 11-15).

Antibodies may be produced by means of phage display according to thefollowing procedure:

The human naive antibody gene libraries HALT/8 were used for theisolation of recombinant single chain F-Variable domains (scFv) againstadrenomedullin peptide. The antibody gene libraries were screened with apanning strategy comprising the use of peptides containing a biotin taglinked via two different spacers to the adrenomedullin peptide sequence.A mix of panning rounds using non-specifically bound antigen andstreptavidin bound antigen were used to minimize background ofnon-specific binders. The eluted phages from the third round of panninghave been used for the generation of monoclonal scFv expressing E. colistrains. Supernatant from the cultivation of these clonal strains hasbeen directly used for an antigen ELISA testing (see Hust, M., Meyer,T., Voedisch, B., Milker, T., Thie, H., El-Ghezal, A., Kirsch, M.I.,Schütte, M., Helmsing, S., Meier, D., Schirrmann, T., Dübel, S., 2011. Ahuman scFv antibody generation pipeline for proteome research. Journalof Biotechnology 152, 159-170; Schütte, M., Thullier, P., Pelat, T.,Wezler, X., Rosenstock, P., Hinz, D., Kirsch, M. I., Hasenberg, M.,Frank, R., Schirrmann, T., Gunzer, M., Hust, M., Dübel, S., 2009.Identification of a putative Crf splice variant and generation ofrecombinant antibodies for the specific detection of Aspergillusfumigatus. PLoS One 4, e6625).

Humanization of murine antibodies may be conducted according to thefollowing procedure: For humanization of an antibody of murine originthe antibody sequence is analyzed for the structural interaction offramework regions (FR) with the complementary determining regions (CDR)and the antigen. Based on structural modeling an appropriate FR of humanorigin is selected and the murine CDR sequences are transplanted intothe human FR. Variations in the amino acid sequence of the CDRs or FRsmay be introduced to regain structural interactions, which wereabolished by the species switch for the FR sequences. This recovery ofstructural interactions may be achieved by random approach using phagedisplay libraries or via directed approach guided by molecular modeling(see Almagro J C, Fransson J., 2008. Humanization of antibodies. FrontBiosci. 2008 Jan. 1; 13:1619-33).

Another subject matter of the present invention is a vasopressor for usein treatment of a subject in need of fluid resuscitation oradministration of a vasopressor wherein said subject is identifiedaccording to any of the above described in vitro methods including allembodiments of said in vitro methods.

FIGURE DESCRIPTION

FIG. 1:

FIG. 1 shows a typical ADM dose/signal curve. And an ADM dose signalcurve in the presence of 100 μg/mL antibody NT-H.

FIG. 2

Predicting in-hospital mortality—Results from logistic regression

FIG. 3

Predicting in-hospital mortality—ADM is independent from Apache andprovides additional prognostic information

FIG. 4

Mean arterial pressure depending on plasma ADM levels. Scatter-plot andcorrelation coefficient are shown for values obtained from patients atadmission. Statistical significance was p<0.0001.

FIG. 5

Adrenomedullin concentrations in patients at admission requiringvasopressor therapy vs patients not requiring vasopressor therapy. Thedifference between the two groups was statistically significant(p<0.0001).

FIG. 6

ADM concentrations of patients at admission, who were treated withvasopressors on admission (“on ADM”), who did not require vasopressortherapy within the first four days after admission (“never”), and whorequired vasopressor therapy within the first four days after admission,but not on the day of admission (“later”). In the graph, the normalrange of ADM concentrations is indicated.

FIG. 7

Receiver Operator Characteristics (ROC) curve for ADM concentrations ofacute heart failure patients requiring (sensitivity) and not requiring(specificity) vasopressor therapy. The area under the curve was 0.75(p<0.0001).

EXAMPLE 1

Generation of Antibodies and Determination of their Affinity Constants

We developed mouse monoclonal antibodies binding to the N-terminal,mid-regional and C-terminal part of ADM and their affinity constantswere determined (table 1).

Peptides for Immunization

Peptides were supplied by JPT Peptide Technologies GmbH (Berlin,Germany). Peptides were coupled to BSA using the Sulfo-SMCC crosslinkingmethod. The crosslinking procedure was performed according themanufacturers instructions (Thermo Fisher/Pierce). The murine antibodieswere generated according to the following method:

A Balb/c mouse was immunized with 100 μg Peptide-BSA-Conjugate at day 0and 14 (emulsified in 100 μl complete Freund's adjuvant) and 50 μg atday 21 and 28 (in 100 μl incomplete Freund's adjuvant). Three daysbefore the fusion experiment was performed, the animal received 50 μg ofthe conjugate dissolved in 100 μl saline, given as one intraperitonealand one intra venous injection.

Splenocytes from the immunized mouse and cells of the myeloma cell lineSP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C.After washing, the cells were seeded in 96-well cell culture plates.Hybrid clones were selected by growing in HAT medium [RPMI 1640 culturemedium supplemented with 20% fetal calf serum and HAT-Supplement]. Aftertwo weeks the HAT medium is replaced with HT Medium for three passagesfollowed by returning to the normal cell culture medium.

The cell culture supernatants were primary screened for antigen specificIgG antibodies three weeks after fusion. The positive testedmicrocultures were transferred into 24-well plates for propagation.After retesting the selected cultures were cloned and recloned using thelimiting-dilution technique and the isotypes were determined.

(Lane, R. D. “A short-duration polyethylene glycol fusiontechnique forincreasing production of monoclonal antibody-secreting hybridomas”, J.Immunol. Meth. 81: 223-228; (1985), Ziegler, B. et al. “Glutamatedecarboxylase (GAD) is not detectable on the surface of rat islet cellsexamined by cytofluorometry and complement-dependent antibody-mediatedcytotoxicity of monoclonal GAD antibodies”, Horm. Metab. Res. 28: 11-15,(1996)).

TABLE 1 ADM Affinity constants Antigen/Immunogen Region Designation Kd(M−1) YRQSMNNFQGLRSFGC  1-16 NT-ADM 1.6 × 10⁹ CTVQKLAHQIYQ 21-32 MR-ADM 2 × 10⁹ CAPRSKISPQGY-NH2 C-42-52 CT-ADM 1.1 × 10⁹

Monoclonal Antibody Production

Antibodies were produced via standard antibody production methods (Marxet al, Monoclonal Antibody Production, ATLA 25, 121, 1997) and purifiedvia Protein A. The antibody purities were >95% based on SDS gelelectrophoresis analysis.

Affinity Constants

To determine the affinity of the antibodies to Adrenomedullin, thekinetics of binding of Adrenomedullin to immobilized antibody wasdetermined by means of label-free surface plasmon resonance using aBiacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany).Reversible immobilization of the antibodies was performed using ananti-mouse Fc antibody covalently coupled in high density to a CMSsensor surface according to the manufacturer's instructions (mouseantibody capture kit; GE Healthcare).

Labelling procedure (tracer): 100 ug (100 ul) of antibody (1 mg/ml inPBS, pH 7.4,) was mixed with 10 ul Akridinium NHS-ester (1 mg/ml inacetonitrile, InVent GmbH, Germany) (57) and incubated for 20 min atroom temperature. Labelled CT-H was purified by Gel-filtration HPLC onBio-Sil® SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purifiedlabeled antibody was diluted in (300 mmol/L potassiumphosphate, 100mmol/L NaCl, 10 mmol/L Na-EDTA, 5 g/L Bovine Serum Albumin, pH 7.0). Thefinal concentration was approx. 800.000 relative light units (RLU) oflabelled compound (approx. 20 nglabeled antibody) per 200 μL.Akridiniumesterchemiluminescence was measured by using an AutoLumat LB953 (Berthold Technologies GmbH & Co. KG).

Solid phase: Polystyrene tubes (Greiner Bio-One International AG,Austria) were coated (18 h at room temperature) with antibody ((1.5 μgantibody/0.3 mL 100 mmol/L NaCl, 50 mmol/L TRIS/HCl, pH 7.8). Afterblocking with 5% bovine serum albumine, the tubes were washed with PBS,pH 7.4 and vacuum dried.

-   -   Calibrators:    -   Synthetic human ADM (Bachem, Switzerland) was linearily diluted        using 50 mMTris/HCl, 250 mMNaCl, 0.2% Triton X-100, 0.5% BSA, 20        tabs/L Protease cOmplete Protease Inhibitor Cocktail Tablets        (Roche AG); pH 7.8. Calibrators were stored at −20° C. before        use.

EXAMPLE 2

Determination of the antibody combination that yields high signal/noiseratios

ADM Immunoassay:

50 ul of sample (or calibrator) was pipetted into coated tubes, afteradding labeleld second antibody (200 ul), the tubes were incubated for 2h at room temperature. Unbound tracer was removed by washing 5 times(each 1 ml) with washing solution (20 mM PBS, pH 7.4, 0.1% TritonX-100).

Tube-bound chemiluminescence was measured by using the LB 953

All antibodies were used in a sandwich immunoassay as coated tube andlabeled antibody and combined in the following variations (table 2):

Incubation was performed as described under hADM-Immunoassay. Resultsare given in ratio of specific signal (at 10 ng/ml ADM)/background(sample without ADM) signal.

TABLE 2 Signal/ NT-ADM MR-ADM CT-ADM noise ratio tracer tracer tracerNT-ADM / 195 241 MRADM 204 / 904 CT-ADM 260 871 /

Surprisingly, we found the combination of MR-ADM and CT-ADM ascombination for highest signal/noise ratio.

Subsequently, we used this antibody-combination for furtherinvestigations. We used MR-ADM as solid phase antibody and CT-ADM aslabeled antibody. A typical dose/signal curve is shown in FIG. 1. Theanalytical sensitivity (average of 10 runs, ADM-free sample+2SD) of theassay was 2 pg ADM/ml.

EXAMPLE 3

Stability of human Adrenomedullin:

Human ADM was diluted in human Citrate plasma (n=5, final concentration10 ng ADM/ml) and incubated at 24° C. At selected time points, aliquotswere frozen at −20° C. Immediately after thawing the samples hADM wasquantified by using the hADM immunoassay described above.

Table 3 shows the stability of hADM in human plasma at 24° C.

Average ADM Relative Time recovery loss of immune Loss of immune (h) (N= 5) reactivity reactivity %/h 0 100 / / 2 99.2 0.8 0.4 4 96.4 3.6 0.8 888.2 11.8 1.5 Average: 0.9%/h

Surprisingly, using the antibody-combinations MR-ADM and CT-ADM in asandwich immune assay, the preanalytical stability of the analyte ishigh (only 0.9%/h average loss of immune reactivity). In contrast, usingother assay methods, a plasma half life of only 22 min was reported(Hinson 2000). Since the time from taking sample to analysis in hospitalroutine is less than 2 h, the used ADM detection method is suitable forroutine diagnosis. It is remarkable, that any non routine additives tosamples (like Aprotinin, (20)) are not needed to reach acceptableADM-immune reactivity stabilities.

Example 4

Reproducibility of calibrator-preparations.

We found a high variation of results, preparing calibrators for ADMassays (average CV 8.5%, see table 4). This may be due to highadsorption of hADM to plastic and glass surfaces (see also (58). Thiseffect was only slightly reduced by adding detergents (up to 1% Triton X100 or 1% Tween 20), protein (up to 5% BSA) and high ionic strength (upto 1M NaCl) or combinations thereof. Surprisingly, if a surplus of antiADM antibody (10 ug/ml) is added to the calibrator dilution buffer, therecovery and reproducibility of ADM assay calibrator-preparations wassubstantially improved to <1% of inter preparation CV (table 4).

Fortunately, the presence of N-terminal antibodies did not affect theADM-signal generated by the combination of MR- and C-terminal antibodies(FIG. 1).

TABLE 4 In the presence Inter Inter of NT-ADM preparation preparationantibody CV Without CV calibrator (10 ug/ml) (%) antibody (%) 100 ng/ml3453 s/n-r 0.9 2842 s/n-r 2.8 10 ng/ml 1946 s/n-r 0.8 1050 s/n-r 7.9 1ng/ml 179 s/n-r 1.1 77 s/n-r 14.8 Average: 0.93 Average: 8.5

Inter preparation variation of calibrators.

ADM assay calibrators were prepared as described above with and without10 ug/ml of NT-ADM-antibody. Coeffients of variation are given from 5independent preparation runs. The calibrators were measured using theADM assay described above. s/n-r=signal to noise ratio.

For all following studies, we used an ADM assay, based on calibrators,prepared in the presence of 10 ug/ml of NT-ADM antibody and 10 ug/ml ofNT-ADM antibody as supplement in the tracer buffer.

EXAMPLE 5

Sensitivity

The goal of assay sensitivity was to completely cover the ADMconcentration of healthy subjects.

ADM concentration in healthy subjects:

Healthy subjects (n=100, average age 56 years) were measured using theADM assay. The median value was 24.7 pg/ml, the lowest value 11 pg/mland the 99^(th) percentile 43 pg/ml. Since the assay sensitivity was 2pg/ml, 100% of all healthy subjects were detectable using the describedADM assay.

A commercial Assay was used to measure MR-proADM (BRAHMS MR-proADMKRYPTOR) (BRAHMS GmbH, Hennigsdorf, Germany) (ClinBiochem. 2009 May;42(7-8):725-8. doi: 10.1016/j.clinbiochem.2009.01.002. Epub 2009 Jan.23.

Homogeneous time-resolved fluoroimmunoassay for the measurement ofmidregionalproadrenomedullin in plasma on the fully automated systemB.R.A.H.M.S KRYPTOR.Caruhel P, Mazier C, Kunde J, Morgenthaler NG,Darbouret B.)

EXAMPLE 6

Clinical Study

101 ED patients fulfilling the definition of sepsis (Dellinger R P, LevyM M, Carlet J M, Bion J, Parker M M, Jaeschke R, Reinhart K, Angus D C,Brun-Buisson C, Beale R et al: Surviving Sepsis Campaign: internationalguidelines for management of severe sepsis and septic shock: 2008.Critical care medicine 2008, 36(1):296-327.) were subsequentlyhospitalized (average 5 days of hospitalization) and received a standardof care treatment. EDTA-plasma was generated from day 1 (EDpresentation) and one sample each day during hospital stay. The time tofreeze samples for later ADM-measurement was less than 4 h. Patientcharacteristics are summarized in table 5

TABLE 5 all in hospitaldeaths discharged Variable (n = 101) (n = 27) (n= 74) p-value Demographics Gender - male 60 (60) 13 (48) 47 (64) 0.163Age - median [IQR] 78 [72-72] 77 [71.25-83] 80 [75-84.5] 0.142Examination variables BP systolic (mmHg) - 115 [100-100] 120[106.25-138.75] 105 [80-120] 0.001 median [IQR] BP diastolic (mmHg) - 65[60-60] 65 [60-85] 60 [50-70] 0.002 median [IQR] HR - median [IQR] 100[94-94] 100 [94-114.75] 100 [93.5-107.5] 0.407 RR - median [IQR] 24[22-22] 24 [22-28] 26 [24-28] 0.069 MAP (mmHg) - 83.3 [74-74] 83.3[77.62-100.75] 81.6 [63.5-89] 0.026 median [IQR] concomitantdiseasesCardiovascular-yes 26 (25.7) 9 (33.3) 17 (23) 0.311 Hypertensive-yes 47(46.5) 13 (48.1) 34 (45.9) 1.000 Diabetes -yes 35 (34.7) 9 (33.3) 26(35.1) 1.000 Cancere-yes 13 (12.9) 3 (11.1) 10 (13.5) 1.000routinelabaratory variables Blood culture-yes 31 (31) 5 (19) 26 (35)0.246 negative 15 (16.3) 2 (8) 13 (19.4) positive 16 (17.4) 3 (12) 13(19.4) Creatinine clearance 48 [23.25-23.25] 56 [29.25-80] 31.5[14.75-66] 0.043 (ml/min) - median [IQR] Creatinine - median [IQR] 1.3[0.9-0.9] 1.25 [0.9-2.08] 1.8 [1-3.15] 0.080 UREA - median [IQR] 36[21-21] 31.5 [20-53.25] 51 [42-87] 0.004 GCS - median [IQR] 15 [10-10]15 [12.5-15] 8 [8-11] <0.001 Pcr - median [IQR] 16 [6.6-6.6] 14.5[6.7-23.7] 17.35 [6.6-28.05] 0.846 Gluco - median [IQR] 113.5[94.5-94.5] 110 [95.5-144] 128 [94-160.5] 0.400 biliru - median [IQR]0.9 [0.71-0.71] 0.9 [0.7-1.03] 0.91 [0.77-1.18] 0.534 GR - median [IQR]3.8 [3.3-3.3] 3.8 [3.2-4.3] 3.7 [3.4-4.2] 0.684 GB - median [IQR] 12700[6774-6774] 13100 [8115-17565] 11920 [25.55-18790] 0.343 PLT - median[IQR] 213 [150-150] 217 [154.75-301] 185 [130-236.5] 0.113 HCT - median[IQR] 32 [28-28] 31.5 [28-37] 34 [31.25-39.5] 0.149 Leuco/Neutr (%) - 87[80-80] 86 [78.25-89.95] 91 [87-93.05] 0.001 median [IQR] HB - median[IQR] 10.4 [9.47-9.47] 10.15 [9.3-12.4] 10.85 [9.9-12.67] 0.220 Na -median [IQR] 137 [134-134] 137 [133-141] 139 [134-144.5] 0.204 K -median [IQR] 3.9 [3.5-3.5] 3.9 [3.6-4.3] 3.9 [3.3-5.1] 0.982 INR -median [IQR] 1.19 [1.1-1.1] 1.19 [1.1-1.4] 1.18 [1.04-1.36] 0.731 TC -median [IQR] 38.4 [36-36] 38.5 [38.12-38.7] 36 [35.55-38.5] <0.001SAO2 - median [IQR] 94 [90-90] 95 [90.25-97] 93 [88.5-95.5] 0.119 pH -median [IQR] 7.45 [7.38-7.38] 7.46 [7.4-7.5] 7.4 [7.24-7.4] <0.001 PO2 -median [IQR] 67 [56-56] 66.5 [56-78] 67 [56.5-79.5] 0.806 PCO2 - median[IQR] 36 [32-32] 37.5 [33-43.75] 34 [30-41] 0.245 Lact - median [IQR]1.5 [1-1] 1.3 [0.83-1.9] 2.5 [1.4-4.15] <0.001 Bic - median [IQR] 23.5[21-21] 24.25 [21.43-28] 21 [17.35-23.25] 0.001 FiO2 (%) - median [IQR]21 [21-21] 21 [21-23.25] 24 [21-45] <0.001 otherAcuteorgandisfunction-yes 39 (43.3) 16 (64) 23 (35.4) 0.021 Apache score(%) - 19 [12.5-12.5] 14.65 [12.12-20.38] 32 [20-39] <0.001 median [IQR]Days hospitalized - 5 [2-2] 6 [4-7] 2 [1-6] 0.003 median [IQR]treatmentatbaseline Diuresis (cc) - 900 [600-600] 1000 [700-1200] 450[200-1025] <0.001 median [IQR] Steroids -yes 16 (15.8) 4 (14.8) 12(16.2) 1.000 Vasopressors-yes 18 (17.8) 13 (48.1) 5 (6.8) <0.001Antibiotics-yes 101 (100) 27 (100) 74 (100) 1.000 Fluid therapy-yes 101(100) 27 (100) 74 (100) 1.000 newbiomarker ADM (pg/mL) - 53.8[37.4-94.0] 93.9 [48.7-241] 50.1 [32.2-77.8] <0.001 median [IQR]MR-proADM 0.54 [0.32-0.86] 0.98 [0.42-18.4] 0.46 [0.28-0.82] <0.001(nmol/L) [IQR]

26.7% of all patients died during hospital stay and are counted astreatment non-responder, 73.3% of all patients survived the sepsis andare counted as treatment responder.

66% off all patients presenting with sepsis had a non-normal ADMvalue >43 pg/ml (99 th percentile), indicating ADM not to be a markerfor the infection.

Results of Clinical Study

Initial ADM is highly prognostic.

We correlated the initial ADM value with the in-hospital mortality andcompared ADM with APACHE 2 score. ADM is highly prognostic for sepsisoutcome (see FIG. 2) and comparable to APACHE 2 score. There is asignificant added information if ADM and APACHE 2 are combined (FIG. 3).

ADM in treatment monitoring.

Patients were treated based on standard of care treatments (table 5).The average hospitalization time was 5 days. ADM was measured each dayin hospital (day 1=admission) and correlated to in hospital mortality(table 6). ADM changed during hospital stay and the change during timeimproved the prognostic value by 52% from initial Chi² of 19.2 to 29.2on day 5.

Using a simple cut off model at 70 pg/ml of ADM showed a 68% risk ofdeath for patients starting at ADM concentrations >70 pg/ml and remainall the hospital stay >70 pg/ml (treatment non-responder). Patientshaving all time an ADM value <70 pg/ml or developing from >70 pg/ml to<70 pg/ml had a mortality of only 11% (well treated/treatment responder)and patients presenting with ADM values >70 pg/ml and reducing their ADMconcentration during hospital treatment to values <70 pg/ml had a 0%mortality. There were no patients developing from <70 pg/ml to >70 pg/mlduring hospital treatment. The average time needed to generateresponder/nonresponder information for all patients was about 1 day.The >70 pg/ml—patients responding to treatment during hospital stayneeded about 2 days to indicate treatment success by ADM.

TABLE 6 Patients Patient all Patient all changed days >70 days <70from >70 pg/ml pg/ml pg/ml to <70 pg/ml N 28/101 73/101 15/73 (27.7%)(72.3%) (20.5%) Mortality 68% 11% 0% Average days after 1 day 1.2 days2.2 days hospitalization of change from ADM >70 pg/ml to ADM <70 pg/mlor no change

Relation of Plasma ADM with Mean Arterial Pressure (MAP) and need forVasopressor Therapy

We found a significant correlation of ADM concentrations with meanarterial pressure (FIG. 4) and with the requirement for vasopressortherapy to treat/prevent shock (FIG. 5).

We also investigated the temporal relationship of ADM concentrations andthe requirement for vasopressor therapy (FIG. 6): Of the 101 patientsinvestigated, already 18 required vasopressor therapy at admission; themedian ADM concentration for these patients at admission was 129 pg/mL.Patients, who never required vasopressor therapy during their hospitalstay within the first four days after admission (n=79), had a median ADMconcentration of 48.5 pg/mL. Importantly, patients, who requiredvasopressor therapy during their hospital stay later than on admissionhad elevated ADM levels already at admission (median 87.2 pg/mL), e.g.:the elevation of plasma ADM concentration was preceding the vasopressortherapy.

In the plasma samples also MR-proADM, a stable fragment of the ADMprecursor molecule, was measured. MR-proADM has been proposed as asurrogate marker for mature ADM release (Struck J, Tao C, Morgenthaler NG, Bergmann A: Identification of an Adrenomedullin precursor fragment inplasma of sepsis patients. Peptides 2004, 25(8):1369-1372, MorgenthalerN G, Struck J, Alonso C, Bergmann A: Measurement of midregionalproadrenomedullin in plasma with an immunoluminometric assay. Clinicalchemistry 2005, 51(10):1823-1829). A commercial MR-proADM Assay was used(BRAHMS MR-proADM KRYPTOR) according to the instructions of themanufacturer (BRAHMS GmbH, Hennigsdorf, Germany). Median levels for theday of admission were 0.63 nmol/L for patients not requiring vasopressortherapy, and 1.57 nmol/L for patients requiring vasopressor therapy.Concentrations of MR-proADM were significantly correlated withconcentrations of ADM (r=0.79).

EXAMPLE 7

Cut off Analysis for Diagnosis and Prediction of Vasopressor need.

Patient data see Example 6. The analysis was performed based on thefirst blood draw taken during emergency unit presentation of the sepsispatient.

A cut off value of 70 pg/ml was selected, <70 pg/ml indicated a low riskof vasopressor need and >70 pg/ml indicated a high risk of vasopressorneed. Group 1 are patients not needed Vasopressors (MAP<=66 mmHg) norreceived Vasopressors at presentation nor during a 4 day follow upperiod. Group 2 are Patients with Vasopressor need (MAP<=66 mmHg) orreceived Vasopressors at presentation. Group 3 are patients withoutVasopressor need nor received Vasopressors at presentation but developedVasopressor need during a 4 day follow up period. Patients (n=2) withmissing information about Vasopressor treatment were excluded.

TABLE 7 <70 pg/ml >70 pg/ml ADM ADM Group 1 56 15 79% specificity Group2 3 18 89.5% sensitivity Group 3 2 5 71% correct classification

Using a simple cut off analysis at 70 pg/ml 89.5% of all patientsneeding vasopressors at ED presention were identified by ADM (group 2).There were 20 patients >70 pg/ml having no vasopressor need atpresentation (group 1/3), 15 (75%) did not developed vasopressor needduring 4 day follow up and 5 (25%) of patients developed Vasopressorneed during a 4 day follow up. In contrast, if ADM was <70pg/ml atpatients without vasopressor need at presentation (Group 1/3), 56(96.5%) did not developed vasopressor need during the 4 day follow upand only 2 (3.5%) developed a vasopressor need. The risk of developingvasopressor need during next 4 days of patients with an ADM value above70 pg/ml is 7.1 times higher than for patients with ADM levels below 70pg/ml (25% vs 3.5%).

Since blood pressure is always monitored, from a clinical point of view,patients with high ADM (>70pg/ml) without vasopressor need atpresentation should be vasopressor treated by adapting the points ofdecision from <66 mmHg MAP to e.g. <75 mmHg aiming earlier support ofcirculation to protect patient from low blood pressure associated organdysfunctions and subsequent high mortality. Using this rule forpatients >70 pg/ml ADM and treating with vasopressors at MAP<=75 mmHg,patients (Group 3) would be treated in average 1,6 days before standardof care treatment (<=66mmHg).

Similar results were obtained, when MR-proADM with a cut-off value of0.78 noml/L was used in the analysis instead of ADM.

TABLE 8 <0.78 noml/L >0.78 noml/L MR-proADM MR-proADM Group 1 54 1776.1% specificity Group 2 4 17 81% sensitivity Group 3 2 5 71% correctclassification

EXAMPLE 8

Clinical Study/Acute Heart Failure

Recruited patients were patients admitted to the emergency departmentwith acute heart failure. Patient characteristics: Mean±SD age 74.3±12.2y; n=1022 (643 male, 63%); previous Ischemic Heart Disease 31%,hypertension 58%, Diabetes 33%, Heart failure 35%. Patients werefollowed up for 2 years. Plasma samples for measurement of ADM and otheranalytes were gained on the day of admission.

Cox analysis revealed that ADM was an independent predictor of 1 yeardeath (table 9) and 1 year death/hospitalization due to acutedecompensated Heart Failure (table 10). Logistic regression analysisrevealed that ADM was an independent predictor of in hospital death(table 11).

Patients who required vasopressor therapy (inotropes) had significantlyhigher ADM concentrations that all other patients (area under thecurve=0.75; p<0001; FIG. 7).

TABLE 9 Hazard Ratio Hazard Ratio (P value) (P value) UnivariateMultivariate Age 1.04 (<0.0005) 1.04 (<0.0005) Past history HF 1.48(<0.001) N.S. Past history 1.79 (<0.0005) N.S. Renal failure Heart rate0.99 (<0.003) N.S. Systolic BP 0.98 (<0.0005) 0.98 (<0.0005) Respiratoryrate 1.02 (<0.0005) 1.02 (<0.001) NYHA 1.61 (<0.0005) N.S. Urea 1.03(<0.0005) 1.05 (<0.0005) Creatinine 1.003 (<0.0005) N.S. Na 0.96(<0.0005) 0.95 (<0.0005) NTproBNP 2.73 (<0.0005) N.S. ADM 3.92 (<0.0005)2.35 (<0.0005)

TABLE 10 Hazard Ratio Hazard Ratio (P value) (P value) UnivariateMultivariate Age 1.02 (<0.0005) 1.02 (<0.001) Past history HF 1.72(<0.001) 1.33 (<0.023) Past history 1.75 (<0.0005) N.S. Renal failureHeart rate 0.99 (<0.002) N.S. Systolic BP 0.99 (<0.0005) 0.99 (<0.012)Respiratory rate 1.01 (<0.008) 1.02 (<0.0005) NYHA 1.53 (<0.0005) 1.29(<0.018) Urea 1.03 (<0.0005) 1.05 (<0.0005) Creatinine 1.002 (<0.0005)N.S. Na 0.98 (<0.012) 0.98 (<0.03) NTproBNP 1.82 (<0.0005) N.S. ADM 2.75(<0.0005) 1.67 (<0.01)

TABLE 11 Odds Ratio Odds Ratio (P value) (P value) UnivariateMultivariate Age 1.04 (<0.001) 1.04 (<0.039) Past history HF 1.98(<0.007) 1.33 (<0.023) Past history 2.71 (<0.0005) N.S. Renal failureHeart rate 0.99 (<0.074) N.S. Systolic BP 0.99 (<0.071) N.S. Respiratoryrate N.S. NYHA 1.67 (<0.023) N.S. Urea 1.078 (<0.0005) 1.069 (<0.015)Creatinine 1.008 (<0.0005) N.S. Na 0.929 (<0.0005) 0.916 ((<0.0005)Troponin I N.S. N.S. NTproBNP 3.22 (<0.001) N.S. ADM 10.75 (<0.0005)5.182 (<0.001)

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Adrenomedullin precursor fragment in plasma of sepsis patients. Peptides2004, 25(8):1369-1372.

1-20. (canceled)
 21. A composition comprising: a complex comprising atleast one binder to proADM (SEQ ID No. 1) and/or fragments and at leastone binder to MR-proADM and/or fragments thereof, sample of bodily fluidobtained from a subject who is not suffering from a physiological shockstate, wherein the complex contains more than 70 pg/ml of proADM (SEQ IDNo. 1) and/or fragments, and/or its isoforms and more than 0.7 nmol/L ofplasma MR-proADM and/or fragments thereof within said composition. 22.The composition of claim 21, wherein said proADM (SEQ ID No. 1) and/orfragments thereof having at least 6 amino acids are selected from thegroup comprising mature ADM (SEQ ID No. 4) and/or mature ADM 1-52-Gly(SEQ ID No. 5) and MR-proADM (SEQ ID No.3) and CT-ADM (SEQ ID No. 6).23. The composition of claim 22 wherein either a level of mature ADM(SEQ ID No. 4) immunoreactivity and/or a level of mature ADM 1-52-Gly(SEQ ID No. 5)—immunoreactivity or a level of MR-proADM (SEQ ID No.3)immunoreactivity or a level of CT-ADM (SEQ ID No.6) immunoreactivity isabove a threshold.
 24. The composition of claim 21, wherein the level ofpro-ADM (SEQ ID No. 1) and/or fragments thereof is measured by using atleast one binder selected from the group consisting of a binder thatbinds to a region comprised within the following sequence of mature ADM(SEQ ID No. 4) and/or mature ADM 1-52-Gly (SEQ ID No. 5) and a secondbinder that binds to a region comprised within the following sequence ofmature ADM (SEQ ID No. 4) and/or mature ADM 1-52-Gly (SEQ ID No. 5). 25.The composition of claim 21, wherein the level of pro-ADM (SEQ ID No. 1)and/or fragments thereof is measured by using at least one binderselected from the group consisting of a binder that binds to a regioncomprised within the following sequence of MR-proADM (SEQ ID No. 3) anda second binder that binds to a region comprised within the followingsequence of MR-proADM (SEQ ID No. 3)
 26. The composition of claim 21,wherein the level of pro-ADM (SEQ ID No. 1) and/or fragments thereof ismeasured by using at least one binder selected from the group consistingof a binder that binds to a region comprised within the followingsequence of CT-proADM (SEQ ID No. 6) and a second binder that binds to aregion comprised within the following sequence of CT-proADM (SEQ ID No.6).
 27. The composition of claim 21, wherein an assay is used formeasuring the level of proADM (SEQ ID No.1) and/or fragments thereofhaving at least 6 amino acids wherein the assay sensitivity of saidassay is able to quantify the ADM of healthy subjects and is <70 pg/ml.28. The composition of claim 21, wherein said binders exhibits a bindingaffinity to proADM (SEQ ID No. 1) and/or fragments thereof of at least10⁷ M⁻¹.
 29. The composition of claim 21, wherein said binders areselected from the group consisting of an antibody or an antibodyfragment and a non-Ig scaffold binding to proADM (SEQ ID No. 1) and/orfragments thereof.
 30. The composition of claim 27, wherein said assayis a sandwich assay.
 31. The composition of claim 21, wherein saidbinders are labeled in order to be detected.
 32. The composition ofclaim 21, wherein at least one of said binders is bound to a solidphase.
 33. The composition of claim 21, wherein said sample of bodilyfluid is selected from the group consisting of human citrate plasma,heparin plasma, EDTA plasma, whole blood, and serum.